Tools for genetic engineering and gene expression control in Novosphingobium aromaticivorans and Rhodobacter sphaeroides

作者：Ashley N. Hall, Benjamin Hall, Kyle J. Kinney, Gabby G. Olsen, Amy B. Banta, Daniel R. Noguera, Timothy J. Donohue, Jason M. Peters · 发表于：Applied and Environmental Microbiology · 年份：2024 · DOI：10.1128/aem.00348-24 · 被引用次数：11 · 研究领域：CRISPR and Genetic Engineering、Microbial Metabolic Engineering and Bioproduction、Biofuel production and bioconversion

ABSTRACT Alphaproteobacteria have a variety of cellular and metabolic features that provide important insights into biological systems and enable biotechnologies. For example, some species are capable of converting plant biomass into valuable biofuels and bioproducts that have the potential to contribute to the sustainable bioeconomy. Among the Alphaproteobacteria, Novosphingobium aromaticivorans , Rhodobacter sphaeroides , and Zymomonas mobilis show promise as organisms that can be engineered to convert extracted plant lignin or sugars into bioproducts and biofuels. Genetic manipulation of these bacteria is needed to introduce engineered pathways and modulate expression of native genes with the goal of enhancing bioproduct output. Although recent work has expanded the genetic toolkit for Z. mobilis , N. aromaticivorans and R. sphaeroides still need facile, reliable approaches to deliver genetic payloads to the genome and to control gene expression. Here, we expand the platform of genetic tools for N. aromaticivorans and R. sphaeroides to address these issues. We demonstrate that Tn 7 transposition is an effective approach for introducing engineered DNA into the chromosome of N. aromaticivorans and R. sphaeroides . We screen a synthetic promoter library to identify isopropyl β-D-1-thiogalactopyranoside-inducible promoters with regulated activity in both organisms (up to ~15-fold induction in N. aromaticivorans and ~5-fold induction in R. sphaeroides ). Combining Tn 7 integrat...